Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Methods for the chemical synthesis of butanols are known. For example, 1-butanol may be produced using the Oxo process, the Reppe process, or the hydrogenation of crotonaldehyde (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). 2-Butanol may be produced using n-butene hydration (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). Additionally, isobutanol may be produced using Oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) or Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A:Chem. 220:215-220 (2004)). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly.
Methods of producing butanol by fermentation are also known, where the most popular process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE processes (Blaschek et al., U.S. Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations, and the pathways and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16:11-16 (1998)). Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. patent application Ser. No. 11/527,995), a 2-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. Patent Application No. 60/796,816), and an isobutanol biosynthetic pathway (Maggio-Hall et al., copending and commonly owned U.S. patent application Ser. No. 11/586,315) have been described. However, biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in the fermentation.
Strains of Clostridium that are tolerant to 1-butanol have been isolated by chemical mutagenesis (Jain et al. U.S. Pat. No. 5,192,673; and Blaschek et al. U.S. Pat. No. 6,358,717), overexpression of certain classes of genes such as those that express stress response proteins (Papoutsakis et al. U.S. Pat. No. 6,960,465; and Tomas et al., Appl. Environ. Microbiol. 69(8):4951-4965 (2003)), and by serial enrichment (Quratulain et al., Folia Microbiologica (Prague) 40(5):467-471 (1995); and Soucaille et al., Current Microbiology 14(5):295-299 (1987)). Desmond et al. (Appl. Environ. Microbiol. 70(10):5929-5936 (2004)) report that overexpression of GroESL, a stress response protein, in Lactococcus lactis and Lactobacillus paracasei produced strains that were able to grow in the presence of 0.5% volume/volume (v/v) [0.4% weight/volume (w/v)] 1-butanol. Additionally, the isolation of 1-butanol tolerant strains from estuary sediment (Sardessai et al., Current Science 82(6):622-623 (2002)) and from activated sludge (Bieszkiewicz et al., Acta Microbiologica Polonica 36(3):259-265 (1987)) have been described. However, for most microorganisms described in the art, growth is totally inhibited at a concentration of less than 2.0% w/v 1-butanol when grown in a liquid medium at 37° C. Moreover, microbial strains that have a tolerance to 2-butanol and isobutanol are not known in the art. Therefore, identification of microorganisms that have a high tolerance to 1-butanol, 2-butanol, and isobutanol would represent an advance in the art.
In addition, 2-butanone and ethanol are valuable compounds that can be produced by fermentation using microorganisms. 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant and activator of oxidative reactions. 2-butanone can be made by omitting the last step of the 2-butanol biosynthetic pathway (Donaldson et al., copending and commonly owned U.S. Patent Application No. 60/796,816). Ethanol is in high demand as an alternative fuel. Genetically modified strains of E. coli have been used as biocatalysts for ethanol production (Underwood et al., (2002) Appl. Environ. Microbiol. 68:6263-6272). A genetically modified strain of Zymomonas mobilis that has improved production of ethanol is described in US 2003/0162271 A1. Identification of microorganisms with improved tolerance to 2-butanone and ethanol would enhance the production of these compounds.
There is a need, therefore, for microbial host strains that are more tolerant to butanols and may be used for the bioproduction of butanols to high titer. The present invention addresses this need through the discovery of butanol tolerant microorganisms and development of methods for their isolation. In addition the discovered microorganisms have increased tolerance to 2-butanone and ethanol.